A composition for treating diabetes or diabesity comprising oxyntomodulin analog

ABSTRACT

Disclosed are a composition for preventing or treating diabetes, diabesity or diabetic complications, containing an oxyntomodulin analog as an active ingredient and a method for treating diabetes, diabesity or diabetic complications, including administering a pharmaceutically effective amount of an oxyntomodulin analog to a subject. The oxyntomodulin analog shows a greater activity to activate a GLP-1 receptor and a glucagon receptor, than native oxyntomodulin. The oxyntomodulin analog induces an expansion of beta-cells and increases insulin secretion, thereby reducing blood glucose levels that were increased due to a high-calorie and high-fat diet. The oxyntomodulin analog induces decreases in a body weight and appetite to improve insulin sensitivity and is useful in maintaining normal blood glucose levels.

TECHNICAL FIELD

The present invention relates to a composition for preventing ortreating diabetes, diabesity or diabetic complications, the compositioncomprising an oxyntomodulin analog as an active ingredient. Moreover,the present invention relates to a method for preventing or treatingdiabetes, diabesity or diabetic complications, the method comprisingadministering a pharmaceutically effective amount of an oxyntomodulinanalog to a subject.

BACKGROUND ART

In recent years, in Korea, the intake of fats from foods has increaseddue to economic growth and the westernization of eating habits, andmetabolic diseases such as hyperlipidemia, obesity, diabetes,hypertension, arteriosclerosis, and fatty liver disease, which arecaused by a lack of exercise, have increased.

Diabetes is a kind of metabolic disease in which insulin secretion isinsufficient or normal functions are not made (DeFronzo, 1988). Diabetesis characterized by increased blood glucose levels that cause variousconditions and syndromes. In the case of diabetes, glucose is excretedwith urine. In recent years, due to an increase in obesity, particularlyabdominal obesity, the incidence of diabetes has explosively increased.

Worldwide, the number of diabetic patients was estimated to be 170million in the year 2000 and expected to reach 370 million in the year2030. However, a recent report showed that the number of diabetesalready reached about 350 million worldwide in the year 2008 (Danaei etal., 2011), and thus it is much larger than expected. It was reportedthat about 80% or more of type 2 diabetic patients were obese, whereasonly less than 10% of obese patients were diabetic (Harris et al.,1987). This relationship between diabetes and obesity is because fattyacids are accumulated in beta-cells or insulin-sensitive tissues such asthe kidneys, the liver or the heart due to irregular secretion ofadipokines and free fatty acids, resulting in lipotoxicity.

If a chronic hyperglycemic condition is not suitably treated, it leadsto various pathological conditions in the body. Typically, it increasesthe risk of retinopathy, renal dysfunction, neuropathy, stroke caused byvascular disorder, kidney or heart diseases, diabetic foot ulcer, andcardiovascular disease. Such complications reduce the quality of life,and eventually reduce the life expectancy of diabetic patients. Thus, toprevent diabetic complications, the effective control of blood glucoselevels is essential.

Current methods that are used to control blood glucose levels includelifestyle modification (diet therapy or exercise therapy) and drugtherapy. However, diet therapy or exercise therapy is difficult tocontrol and implement strictly, and the therapeutic effect thereof isalso insufficient. Thus, most diabetic patients rely on lifestylemodification together with the control of blood glucose levels by drugssuch as insulin, insulin secretion stimulators, insulin sensitivityenhancers, and blood glucose level lowering agents.

Insulin that is produced by recombination methods is an essential drugfor type 1 diabetic patients and type 2 diabetic patients whose bloodglucose levels are not controlled, and it is advantageous forcontrolling blood glucose levels. However, it has shortcomings,including a fearful feeling for hypodermic needles, difficulty inadministration, risk of hypoglycaemia, and an increase in weight.

Meglitinides that are insulin secretion stimulators are drugs having avery quick effect, are taken before meals, and include NovoNorm(repaglinide), Fastic (nateglinide), Glufast (mitiglinide), etc. Insulinsensitivity enhancers are characterized in that they cause little or nohypoglycaemia when being taken alone, and examples thereof includemetformin that is a biguanide drug, thiazolidinedione drugs such asAvandia (rosiglitazone), Actos (pioglitazone), etc.

Drugs that were recently developed include GLP-1 agonists developedbased on the action of glucagon-like peptide-1, a hormone thatstimulates insulin secretion, and examples of the GLP-1 agonists includeexenatide and liraglutide. In addition, DPP-4 inhibitors are alsorecently developed new drugs, which inhibit the activity of DPP-4(dipeptidyl peptidase-4), an enzyme that rapidly inactivates GLP-1, andtypical examples thereof include Januvia (sitagliptin). However, thesedrugs were reported to have side effects, including hepatotoxicity,gastrointestinal disorder, cardiovascular disease and carcinogenesis,and the annual cost for treatment of diabetes is also high, and thus isan obstacle in the treatment of diabetes. Indeed, the cost associatedwith pre-diabetes and diabetes reached about 200 trillion Won in the USAin the year 2007 (Dall et al., 2010), and the cost associated withobesity also reached 150 trillion Won in the USA in the year 2008(Finkelstein et al., 2009).

Thus, there is an urgent need for the development of drugs, which can beused to treat both diabetes and diabesity by reducing weight andeffectively lowering blood glucose levels and, at the same time, andhave less side effects.

As a candidate for such drugs, oxyntomodulin has recently receivedattention. Oxyntomodulin is produced from pre-glucagon, a precursor, andis a peptide that can bind to both glucagon-like peptide-1 (GLP-1) andglucagon receptor to perform dual function. Because of suchcharacteristics, oxyntomodulin has been studied for various purposes,including the treatment of obesity, diabetes, hyperlipidemia and fattyliver disease.

However, oxyntomodulin has a problem in that it should be administeredat a high dose, because it has a short half-life in vivo and theactivity thereof is insufficient for use in the treatment of obesity,diabetes, hyperlipidemia and fatty liver disease.

DISCLOSURE Technical Problem

The present inventors have developed an oxyntomodulin analog havingincreased activity compared to native oxyntomodulin and have found thatthe oxyntomodulin analog reduces blood glucose levels, improve glucosetolerance and increases the ratio of glycated hemoglobin (HbA1c) in ahigh-fat diet-induced (HF DIO) mouse model and a diabetic mouse (db/db)model induced by a mutation in the leptin receptor, indicating that theoxyntomodulin analog can be effectively used for the treatment ofdiabetes, diabesity and diabetic complications, thereby completing thepresent invention.

Technical Solution

It is an object of the present invention to provide a composition forpreventing or treating diabetes, diabesity and diabetic complications,comprising an oxyntomodulin analog as an active ingredient.

Another object of the present invention is to provide a method forpreventing or treating diabetes, diabesity and diabetic complications,comprising administering a pharmaceutically effective amount of anoxyntomodulin analog to a subject.

Still another object of the present invention is to provide the use ofthe oxyntomodulin analog of the present invention in the preparation ofa medicament for preventing or treating diabetes, diabesity and diabeticcomplications.

Advantageous Effects

The oxyntomodulin analog of the present invention has a high activity toactivate GLP-1 receptor and glucagon receptor compared to nativeoxyntomodulin. Further, the oxyntomodulin analog of the presentinvention induces the expansion of beta-cells and increases insulinsecretion, thereby reducing blood glucose levels that were increased bya high-calorie and high-fat diet. In addition, the oxyntomodulin analoginduces decreases in body weight and diet intake to improve insulinsensitivity and allow blood glucose levels, which are not controlled dueto insulin resistance, to be maintained at normal levels. Thus, theoxyntomodulin analog can be effectively used for the prevention ortreatment of diabetes and related diseases.

DESCRIPTION OF DRAWINGS

FIG. 1 is a graphic diagram showing the change in body weight caused byadministration of a long-acting oxyntomodulin analog in mice withobesity induced by high-fat diet for a long period of time (26 weeks).The change in body weight was expressed as a percentage relative to thebody weight measured at day 0.

FIG. 2 is a graphic diagram showing an AUC (area under curve) for thechange in blood glucose level caused by administration of a long-actingoxyntomodulin analog in mice with obesity induced by high-fat diet for along period of time (26 weeks).

FIG. 3 is a graphic diagram showing the 4-week change in body weightcaused by 4-week administration of a long-acting oxyntomodulin analog ina mouse model with diabetes induced by a mutation in the leptinreceptor.

FIG. 4 is a graphic diagram showing an AUC (area under curve) for thechange in blood glucose level caused by 4-week administration of along-acting oxyntomodulin analog in a mouse model with diabetes inducedby a mutation in the leptin receptor.

BEST MODE

In one aspect, the present invention provides a composition forpreventing or treating diabetes, diabesity and diabetic complications,comprising an oxyntomodulin analog as an active ingredient.

As used herein, the term “oxyntomodulin” refers to a peptide producedfrom pre-glucagon that is a precursor of glucagon. In the presentinvention, oxyntomodulin is meant to include native oxyntomodulin andits precursor, analog, fragments and variants. Preferably, oxyntomodulinhas an amino acid sequence of SEQ ID NO: 1(HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA).

As used herein, the term “oxyntomodulin variant” is a peptide that hasone or more amino acid residues different from those of the amino acidsequence of native oxyntomodulin and possesses a function of activatingGLP-1 and glucagon receptors. The oxyntomodulin variant can be preparedby any one of substitution, addition, deletion, modification, or acombination thereof of some amino acids of native oxyntomodulin.

As used herein, the term “oxyntomodulin analog” refers to a peptide,peptide derivative or peptide mimic, which is prepared by the addition,deletion or substitution of some amino acids of native oxyntomodulin andcan highly activate both GLP-1 receptor and glucagon receptor, comparedto native oxyntomodulin.

As used herein, the term “oxyntomodulin fragment” refers to a fragmenthaving an addition or deletion of one or more amino acids at the aminoor carboxyl terminal end of native oxyntomodulin, in which the addedamino acids may also be non-naturally occurring amino acids (e.g.,D-type amino acid). This oxyntomodulin fragments has a function ofregulating blood glucose levels in vivo.

Methods for preparing the oxyntomodulin variant, analog and fragment maybe used alone or in combination. For example, the present inventionincludes a peptide, which has one or more amino acids different fromthose of native peptide, has deaminated amino acid residues at theN-terminus and has a function of activating both GLP-1 receptor andglucagon receptor.

Amino acids mentioned herein are abbreviated according to thenomenclature rules of IUPAC-IUB as follows:

Alanine A; Arginine R; Asparagine N;

Aspartic acid D;

Cysteine C;

Glutamic acid E;

Glutamine Q; Glycine G; Histidine H; Isoleucine I; Leucine L; Lysine K;Methionine M; Phenylalanine F Proline P; Serine S; Threonine T;Tryptophan W; Tyrosine Y; Valine V.

In the present invention, the oxyntomodulin analog encompasses anypeptide that is prepared by the substitution, addition, deletion orpost-translational modification (e.g., methylation, acylation,ubiquitination, or intramolecular covalent bonding) of amino acids inthe amino acid sequence of SEQ ID NO: 1 and can activate both theglucagon and GLP-1 receptors. Upon substitution or addition of aminoacids, not only 20 amino acids commonly found in human proteins, butalso atypical or non-naturally occurring amino acids can be used.Commercial sources of atypical amino acids include Sigma-Aldrich,ChemPep Inc., and Genzyme Pharmaceuticals. The peptides including theseamino acids and atypical peptide sequences may be synthesized andpurchased from commercial suppliers, for example, American PeptideCompany or Bachem (USA) or Anygen (Korea).

In a specific embodiment of the present invention, the oxyntomodulinanalog of the present invention is a novel peptide including the aminoacid sequence of the following formula 1:

R1-X1-X2-GTFTSDX3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-R2  Formula 1

whereinR1 is histidine, desamino-histidyl,dimethyl-histidyl(N-dimethyl-histidyl), beta-hydroxyimidazopropionyl,4-imidazoacetyl, beta-carboxy imidazopropionyl or tyrosine;X1 is Aib (aminosiobutyric acid), d-alanine, glycine, Sar(N-methylglycine), serine or d-serine;X2 is glutamic acid or glutamine;X3 is leucine or tyrosine;X4 is serine or alanine;X5 is lysine or arginine;X6 is glutamine or tyrosine;X7 is leucine or methionine;X8 is aspartic acid or glutamic acid;X9 is glutamic acid, serine or alpha-methyl-glutamic acid or is deleted;X10 is glutamine, glutamic acid, lysine, arginine or serine or isdeleted;X11 is alanine, arginine or valine or is deleted;X12 is alanine, arginine, serine or valine or is deleted;X13 is lysine, glutamine, arginine or alpha-methyl-glutamic acid or isdeleted;X14 is aspartic acid, glutamic acid or leucine or is deleted;X15 is phenylalanine or is deleted;X16 is isoleucine or valine or is deleted;X17 is alanine, cysteine, glutamic acid, lysine, glutamine oralpha-methyl-glutamic acid or is deleted;X18 is tryptophan or is deleted;X19 is alanine, isoleucine, leucine, serine or valine or is deleted;X20 is alanine, lysine, methionine, glutamine or arginine or is deleted;X21 is asparagine or is deleted;X22 is alanine, glycine or threonine or is deleted;X23 is cysteine or lysine or is deleted;X24 is a peptide having 2 to 10 amino acids consisting of a combinationof alanine, glycine and serine or is deleted; andR2 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36), GPSSGAPPPSK(SEQ ID NO: 37), HSQGTFTSDYSKYLD (SEQ ID NO: 38), HSQGTFTSDYSRYLDK (SEQID NO: 39), HGEGTFTSDLSKQMEEEAVK (SEQ ID NO: 40) or is deleted (with theexception of the case in which the amino acid sequence of formula 1 isidentical to that of SEQ ID NO: 1).

In order to increase the activity of wild-type oxyntomodulin for theglucagon receptor and the GLP-1 receptor, the oxyntomodulin analog ofthe present invention may be substituted with 4-imidazoacetyl obtainedby deletion of the alpha carbon of histidine at position 1 of the aminoacid sequence of SEQ ID NO: 1, desamino-histidyl obtained by deletion ofthe N-terminal amino group, dimethyl-histidyl(N-dimethyl-histidyl)obtained by modification of the N-terminal amino group with two methylgroups, beta-hydroxy imidazopropionyl obtained by substitution of theN-terminal amino group with a hydroxyl group, or beta-carboxyimidazopropionyl obtained by substitution of the N-terminal amino groupwith a carboxyl group. In addition, the GLP-1 receptor-binding regionmay be substituted with amino acids that enhance hydrophobic and ionicbonds or a combination thereof. Further, a portion of the oxyntomodulinsequence may be substituted with the amino acid sequence of GLP-1 orExendin-4 to increase the activity of the GLP-1 receptor.

Moreover, a portion of the oxyntomodulin sequence may be substitutedwith a sequence that enhances alpha helix. Preferably, amino acids atpositions 10, 14, 16, 20, 24 and 28 of the amino acid sequence offormula 1 may be substituted with amino acids or amino acid derivativesconsisting of Tyr(4-Me), Phe, Phe(4-Me), Phe(4-Cl), Phe(4-CN),Phe(4-NO₂), Phe(4-NH₂), Phg, Pal, Nal, Ala(2-thienyl) andAla(benzothienyl) that are known to stabilize alpha helix, and the typeand number of alpha helix-stabilizing amino acid or amino acidderivatives to be inserted are not limited. Preferably, amino acids atpositions 10 and 14, 12 and 16, 16 and 20, 20 and 24, and 24 and 28 ofthe amino acid sequence may also be substituted with glutamic acid orlysine so as to form rings, and the number of rings to be inserted isnot limited. Most preferably, the oxyntomodulin analog may have an aminoacid sequence selected from among the following formulas 2 to 6.

In a specific embodiment, the oxyntomodulin analog of the presentinvention is a novel peptide including the amino acid sequence of thefollowing formula 2, obtained by substitution of the amino acid sequenceof oxyntomodulin with that of exendin or GLP-1:

R1-A-R3  Formula 2

In another specific embodiment, the oxyntomodulin analog of the presentinvention is a novel peptide including the amino acid sequence of thefollowing formula 3, which is prepared by linking a portion of the aminoacid sequence of oxyntomodulin and a portion of the amino acid sequenceof exendin or GLP-1 via a proper amino acid linker:

R1-B-C-R4  Formula 3

In still another specific embodiment, the oxyntomodulin analog of thepresent invention is a novel peptide including the amino acid sequenceof the following formula 4, wherein a portion of the amino acid sequenceof oxyntomodulin is substituted with an amino acid that enhances thehydrophobic binding to GLP-1 receptor. For example, it is a peptidewherein Leu at position 26 is substituted with the amino acid Ile or Valthat increases hydrophobicity.

R1-SQGTFTSDYSKYLD-D1-D2-D3-D4-D5-LFVQW-D6-D7-N-D8-R3  Formula 4

In still another specific embodiment, the oxyntomodulin analog of thepresent invention is a novel peptide including the amino acid sequenceof the following formula 5, wherein a portion of the amino acid sequenceof native oxyntomodulin is deleted, added, or substituted with otheramino acids in order to increase the abilities of native oxyntomodulinto activate GLP-1 receptor and glucagon receptor:

R1-E1-QGTFTSDYSKYLD-E2-E3-RA-E4-E5-FV-E6-WLMNT-E7-R5  Formula 5

In formulas 2 to 5, R1 is as described in formula 1;

A is selected from the group consisting of SQGTFTSDYSKYLDSRRAQDFVQWLMNT(SEQ ID NO: 41), SQGTFTSDYSKYLDEEAVRLFIEWLMNT (SEQ ID NO: 42),SQGTFTSDYSKYLDERRAQDFVAWLKNT (SEQ ID NO: 43),GQGTFTSDYSRYLEEEAVRLFIEWLKNG (SEQ ID NO: 44),GQGTFTSDYSRQMEEEAVRLFIEWLKNG (SEQ ID NO: 45), GEGTFTSDLSRQMEEEAVRLFIEWAA(SEQ ID NO: 46), and SQGTFTSDYSRQMEEEAVRLFIEWLMNG (SEQ ID NO: 47); B isselected from the group consisting of SQGTFTSDYSKYLDSRRAQDFVQWLMNT (SEQID NO: 41), SQGTFTSDYSKYLDEEAVRLFIEWLMNT (SEQ ID NO: 42),SQGTFTSDYSKYLDERRAQDFVAWLKNT (SEQ ID NO: 43),GQGTFTSDYSRYLEEEAVRLFIEWLKNG (SEQ ID NO: 44),GQGTFTSDYSRQMEEEAVRLFIEWLKNG (SEQ ID NO: 45), GEGTFTSDLSRQMEEEAVRLFIEWAA(SEQ ID NO: 46), SQGTFTSDYSRQMEEEAVRLFIEWLMNG (SEQ ID NO: 47),GEGTFTSDLSRQMEEEAVRLFIEW (SEQ ID NO: 48), and SQGTFTSDYSRYLD (SEQ ID NO:49);

C is a peptide having 2 to 10 amino acids consisting of a combination ofalanine, glycine and serine;D1 is serine, glutamic acid or arginine;D2 is arginine, glutamic acid or serine;D3 is arginine, alanine or valine;D4 is arginine, valine or serine;D5 is glutamine, arginine or lysine;D6 is isoleucine, valine or serine;D7 is methionine, arginine or glutamine;D8 is threonine, glycine or alanine;E1 is serine, Aib, Sar, d-alanine or d-serine;E2 is serine or glutamic acid;E3 is arginine or lysine;E4 is glutamine or lysine;E5 is aspartic acid or glutamic acid;E6 is glutamine, cysteine or lysine;E7 is cysteine or lysine or is deleted;

R3 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36) orGPSSGAPPPSK (SEQ ID NO: 37); R4 is HSQGTFTSDYSKYLD (SEQ ID NO: 38),HSQGTFTSDYSRYLDK (SEQ ID NO: 39) or HGEGTFTSDLSKQMEEEAVK (SEQ ID NO:40); and

R5 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36) orGPSSGAPPPSK (SEQ ID NO: 37) or is deleted (with the exception of thecase in which the amino acid sequences of formulas 2 to 5 are identicalto that of SEQ ID NO: 1).

Preferably, the oxyntomodulin analog of the present invention may be anovel peptide of the following formula 6:

R1-X1-X2-GTFTSDX3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-X22-X23-X24-R2  Formula 6

wherein R1 is histidine, desamino-histidyl, 4-imidazoacetyl or tyrosine;X1 is Aib(aminosiobutyric acid), glycine, serine or d-serine;X2 is glutamic acid or glutamine;X3 is leucine or tyrosine;X4 is serine or alanine;X5 is lysine or arginine;X6 is glutamine or tyrosine;X7 is leucine or methionine;X8 is aspartic acid or glutamic acid;X9 is glutamic acid or alpha-methyl-glutamic acid or is deleted;X10 is glutamine, glutamic acid, lysine or arginine or is deleted;X11 is alanine or arginine or is deleted;X12 is alanine or valine or is deleted;X13 is lysine, glutamine, arginine or alpha-methyl-glutamic acid or isdeleted;X14 is aspartic acid, glutamic acid or leucine or is deleted;X15 is phenylalanine or is deleted;X16 is isoleucine or valine or is deleted;X17 is alanine, cysteine, glutamic acid, glutamine oralpha-methyl-glutamic acid or is deleted;X18 is tryptophan or is deleted;X19 is alanine, isoleucine, leucine or valine or is deleted;X20 is alanine, lysine, methionine or arginine or is deleted;X21 is asparagine or is deleted;X22 is threonine or is deleted;X23 is cysteine, lysine or is deleted;X24 is a peptide having 2 to 10 amino acids consisting of glycine or isdeleted; andR2 is KRNRNNIA (SEQ ID NO: 35), GPSSGAPPPS (SEQ ID NO: 36), GPSSGAPPPSK(SEQ ID NO: 37), HSQGTFTSDYSKYLD (SEQ ID NO: 38), HSQGTFTSDYSRYLDK (SEQID NO: 39) or HGEGTFTSDLSKQMEEEAVK (SEQ ID NO: 40) or is deleted (withthe exception of the case in which the amino acid sequence of formula 6is identical to that of SEQ ID NO: 1). More preferably, theoxyntomodulin analog of the present invention may be selected from thegroup consisting of the peptides of SEQ ID NOs: 2 to 34. Even morepreferably, the oxyntomodulin analog of the present invention may be anoxyntomodulin analog described in Table 1 of Example 2-1.

In an example of the present invention, oxyntomodulin analogs having theamino acid sequences of SEQ ID NOs: 2 to 34, respectively, wereprepared, and it was found that the oxyntomodulin analogs showed anexcellent ability to activate GLP-1 receptor and glucagon receptorcompared to native oxyntomodulin (Example 2). In other words, it couldbe seen from the above results that the oxyntomodulin analog of thepresent invention exhibited excellent effects on the prevention ortreatment of diabetes, diabesity and/or diabetic complications comparedto conventional oxyntomodulin by activating the GLP-1 receptor and theglucagon receptor.

The oxyntomodulin analogs of the present invention are present in theform of conjugates comprising various polymer in order to improve thetherapeutic effect and in vivo half-life of the analogs.

The conjugates of the present invention have longer-acting effects thannative oxyntomodulin, and the long-acting conjugates include anoxyntomodulin prepared by the modification, substitution, addition ordeletion of the amino acids of native oxyntomodulin, an oxyntomodulinconjugated to a biodegradable polymer such as polyethylene glycol (PEG),an oxyntomodulin conjugated to a albumin, antibody, elastin, fibronectinor polysaccharide such as chitin or to a long-acting protein such as animmunoglobulin fragment, an oxyntomodulin conjugated to fatty acidhaving the ability of binding to albumin in vivo, or an oxyntomodulinencapsulated in biodegradable nanoparticles, and the type of long-actingconjugate that is used in the present invention is not limited.

Preferably, the conjugate is a conjugate wherein an oxyntomodulin analoghaving an amino acid sequence selected from the group consisting of theamino acid sequences of SEQ ID NOS: 2 to 34 is linked to animmunoglobulin Fc region through a non-peptidyl polymer.

The immunoglobulin Fc region is a biodegradable polypeptide that ismetabolized in vivo, and thus is safe for use as a carrier for a drug.The immunoglobulin Fc region has a low molecular weight compared to theentire immunoglobulin molecule, and thus is advantageous in terms of thepreparation, purification and yield of conjugates. In addition, becausethe amino acid sequence differs between antibodies, a Fab portionshowing high non-homogeneity is removed, and thus the homogeneity of thematerial can be greatly increased and the possibility of inducing bloodantigenicity can also be reduced.

As used herein, the term “immunoglobulin Fc region” refers to a proteinthat contains the heavy-chain constant region 2 (CH2) and heavy-chainconstant region 3 (CH3) of an immunoglobulin, excluding the heavy-chainand light-chain variable regions, the heavy-chain constant region 1(CH1) and the light-chain constant region 1 (CL1) of the immunoglobulin.It may further include a hinge region at the heavy-chain constantregion. Also, the immunoglobulin Fc region of the present invention maybe an expanded Fc region including part or all of the heavy-chainconstant region 1 (CH1) and/or the light-chain constant region 1 (CL1),except for the heavy-chain and light-chain variable regions, as long asit has an effect that is substantially equal to or better than thenative protein. Further, the immunoglobulin Fc region may be a regionhaving a deletion of a portion of a relatively long amino acid sequencecorresponding to CH2 and/or CH3. Specifically, the immunoglobulin Fcregion of the present invention may comprise 1) a CH1 domain, a CH2domain, a CH3 domain and a CH4 domain, 2) a CH1 domain and a CH2 domain,3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) acombination of one or more domains and an immunoglobulin hinge region(or a portion of the hinge region), or 6) a dimer of each domain of theheavy-chain constant regions and the light-chain constant region.

The immunoglobulin Fc region of the present invention includes a nativeamino acid sequence, and a sequence derivative (mutant) thereof. As usedherein, the term “amino acid sequence derivative” refers to a sequencethat is different from the native amino acid sequence due to thedeletion, insertion, non-conservative or conservative substitution or acombination thereof of one or more amino acid residues of the nativeamino acid sequence. For example, in the case of an IgG Fc, amino acidresidues at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331,which are known to be important in binding, may be used as suitablesites for modification.

In addition, other various derivatives are possible, including one thathas a deletion of a region capable of forming a disulfide bond, or adeletion of some amino acid residues at the N-terminal end of native Fcor an addition of a methionine residue at the N-terminal end of nativeFc. Further, to remove effector functions, a deletion may occur in acomplement-binding site, such as a C1q-binding site and an ADCC(antibody dependent cell mediated cytotoxicity) site. Techniques ofpreparing such sequence derivatives of the immunoglobulin Fc region aredisclosed in International Patent Publication Nos. WO 97/34631, WO96/32478, etc.

Amino acid exchanges in proteins and peptides, which do not generallyalter the activity of the proteins or peptides, are known in the art (H.Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). Themost commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu,Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro,Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, in bothdirections. In addition, the Fc region may, if necessary, be modified byphosphorylation, sulfation, acrylation, glycosylation, methylation,farnesylation, acetylation, amidation, and the like.

The above-described Fc derivatives show biological activity identical tothat of the Fc region of the present invention or have increasedstructural stability against heat, pH, or the like.

In addition, this Fc region may be obtained from native forms isolatedfrom humans and other animals including cows, goats, pigs, mice,rabbits, hamsters, rats and guinea pigs, or may be recombinants orderivatives thereof, obtained from transformed animal cells ormicroorganisms. Herein, the Fc region may be obtained from a nativeimmunoglobulin by isolating a whole immunoglobulin from a living humanor animal body and treating the isolated immunoglobulin with proteinase.When the whole immunoglobulin is treated with papain, it is cleaved intoFab and Fc regions, and when the whole immunoglobulin is treated withpepsin, it is cleaved into pF′c and F(ab)₂ fragments. Fc or pF′c can beisolated using size exclusion chromatography or the like. Preferably, ahuman-derived Fc region is a recombinant immunoglobulin Fc regionobtained from a microorganism.

In addition, the immunoglobulin Fc region may be in the form of havingnative sugar chains or increased or decreased sugar chains compared to anative form, or may be in a deglycosylated form. The increase, decreaseor removal of the immunoglobulin Fc sugar chains may be achieved byconventional methods such as a chemical method, an enzymatic method anda genetic engineering method using a microorganism. The Fc regionobtained by removal of sugar chains from Fc shows a significant decreasein binding affinity to the C1q part and a decrease or loss inantibody-dependent cell-mediated cytotoxicity or complement-dependentcytotoxicity, and thus does not induce unnecessary immune responses invivo. In this regard, an immunoglobulin Fc region in a deglycosylated oraglycosylated form may be more suitable to the object of the presentinvention as a drug carrier.

As used herein, the term “deglycosylation” refers to enzymaticallyremoving sugar moieties from an Fc region, and the term “aglycosylation”refers to an unglycosylated Fc region produced in a prokaryote,preferably E. coli.

Meanwhile, the immunoglobulin Fc region may be derived from humans orother animals including cows, goats, pigs, mice, rabbits, hamsters, ratsand guinea pigs. Preferably, it is derived from humans.

In addition, the immunoglobulin Fc region may be derived from IgG, IgA,IgD, IgE, IgM, or a combination or hybrid thereof. Preferably, it isderived from IgG or IgM, which are among the most abundant proteins inhuman blood, and most preferably from IgG known to enhance thehalf-lives of ligand-binding proteins.

As used herein, the term “combination” means that polypeptides encodingsingle-chain immunoglobulin Fc regions of the same origin are linked toa single-chain polypeptide of a different origin to form a dimer ormultimer. Specifically, a dimer or multimer may be formed from two ormore fragments selected from the group consisting of IgG Fc, IgA Fc, IgMFc, IgD Fc, and IgE Fc fragments.

As used herein, the term “hybrid” means that sequences corresponding totwo or more immunoglobulin Fc fragments of different origins are presentin a single-chain immunoglobulin Fc region. In the present invention,various forms of hybrid are possible. In other words, a hybrid composedof 1 to 4 domains selected from the group consisting of the CH1, CH2,CH3 and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc is possible,and it may include a hinge. Meanwhile, IgG can also be sub-classifiedinto IgG1, IgG2, IgG3 and IgG4, and in the present invention, acombination or hybrid of these subclasses is also possible. Preferably,IgG is the IgG2 ad IgG4 subclass, and most preferably, it is the Fcregion of IgG4 that substantially lacks effector functions such ascomplement-dependent cytotoxicity (CDC).

In other words, the most preferred immunoglobulin Fc region that is usedas a drug carrier in the present invention is an Fc region derived fromhuman IgG4. A human-derived Fc region is more preferable than anon-human-derived Fc region, which may act as an antigen in the humanbody and cause undesirable immune responses such as the production of anew antibody against the antigen.

As used herein, the term on-peptidyl polymer? refers to a biocompatiblepolymer including two or more repeating units linked to each other byany covalent bond in place of a peptide bond. In the present invention,the non-peptidyl polymer may be used interchangeably with thenon-peptidyl linker.

The non-peptidyl polymer that can be used in the present invention maybe selected from the group consisting of polyethylene glycol,polypropylene glycol, an ethylene glycol/propylene glycol copolymer,polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran,polyvinyl ethyl ether, biodegradable polymers such as PLA (poly(lacticacid)) and PLGA (polylactic-glycolic acid), lipid polymers, chitins,hyaluronic acid, and combinations thereof. Preferably, the non-peptidylpolymer is polyethylene glycol. In addition, derivatives thereof knownin the art and derivatives that may be easily prepared by a method knownin the art also fall within the scope of the present invention.

The peptide linker that is used in a fusion protein obtained by aconventional inframe fusion method has drawbacks in that it is easilycleaved by proteinase in vivo, and thus a sufficient effect ofincreasing the serum half-life of the active drug by a carrier cannot beobtained as expected. However, in the present invention, the polymerhaving resistance to proteinase can be used to maintain the serumhalf-life of the peptide, similar to the carrier. Therefore, anynon-peptidyl polymer can be used without limitation in the presentinvention, as long as it is a polymer having the aforementionedfunction, that is, a polymer having resistance to proteinase in vivo.The non-peptidyl polymer has a molecular weight in the range of 1 to 100kDa, and preferably 1 to 20 kDa. The non-peptidyl polymer of the presentinvention, which is linked to the immunoglobulin Fc region, may be onekind of polymer or a combination of different polymers.

The non-peptidyl polymer that is used in the present invention may havea reactive group capable of binding to the immunoglobulin Fc region andthe protein drug. The reactive group at both ends of the non-peptidylpolymer is preferably selected from the group consisting of a reactivealdehyde group, a propionaldehyde group, a butyraldehyde group, amaleimide group and a succinimide derivative.

The succinimide derivative may be succinimidyl propionate, hydroxysuccinimidyl, succinimidyl carboxymethyl, or succinimidyl carbonate. Inparticular, when the non-peptidyl polymer has a reactive aldehyde groupat both ends thereof, non-specific reactions can be minimized, and aphysiologically active polypeptide and an immunoglobulin can beeffectively bound to both ends of the non-peptidyl polymer,respectively. A final product generated by reductive alkylation with analdehyde bond is much more stable than that linked by an amide bond. Thealdehyde reactive group selectively binds to an N-terminus at a low pHand can form a covalent bond with a lysine residue at a high pH such aspH 9.0.

The reactive groups at both ends of the linker that is the non-peptidylpolymer may be the same or different. For example, the non-peptidylpolymer may possess a maleimide group at one end, and an aldehyde group,a propionaldehyde group or a butyraldehyde group at the other end. Whena polyethylene glycol having a reactive hydroxy group at both endsthereof is used as the non-peptidyl polymer, the hydroxy group may beactivated to various reactive groups by known chemical reactions, or apolyethylene glycol having a commercially available modified reactivegroup may be used so as to prepare the long-acting conjugate of thepresent invention. The conjugate of the present invention may be one inwhich each end of the non-peptidyl polymer is linked to theimmunoglobulin Fc region and the amine or thiol group of theoxyntomodulin analog, respectively.

Meanwhile, in the present invention, both ends of the non-peptidylpolymer include reactive groups to which an immunoglobulin Fc region anda protein drug can bind. Examples of the reactive groups include, butare not limited to, an aldehyde group, a propionaldehyde group or abutyraldehyde group, a maleimide group, a succinimide derivative(succinimidyl propionate, hydroxyl succinimidyl, succinimidyl propionatecarboxymethyl or succinimidyl carbonate) and the like.

The reactive groups at both ends of the linker that is the non-peptidylpolymer may be the same or different. For example, the non-peptidylpolymer may have a maleimide group at one end and an aldehyde group, apropionaldehyde group or a butyraldehyde group at the other end. Forexample, when the non-peptidyl polymer has a reactive aldehyde group atone end and a reactive maleimide group at the other end, non-specificreactions can be minimized, and a physiologically active polypeptide andan immunoglobulin can be effectively bound to both ends of thenon-peptidyl polymer. In an example of the present invention, aconjugate was synthesized by linking oxyntomodulin or its analog to theimmunoglobulin Fc region via a covalent bond using the non-peptidylpolymer PEG including a propionaldehyde group alone or both a maleimidegroup and an aldehyde group.

The pharmaceutical composition of the present invention can be used forthe prevention or treatment of diabetes, diabesity and/or diabeticcomplications.

As used herein, the term “prevention” refers to all actions that inhibitor delay the development of a target disease. Specifically, the term“prevention” means administering the oxyntomodulin analog of the presentinvention to control blood glucose levels to normal levels to therebyinhibit or delay the development of diabetes, diabesity or diabeticcomplications.

As used herein, the term “treatment” refers to all actions thatalleviate, ameliorate or relieve the symptoms of the disease developed.Specifically, the term “treatment” means administering the oxyntomodulinanalog of the present invention to maintain blood glucose levels stablyat normal levels to thereby alleviate, ameliorate or relieve theconditions of diabetes, diabesity or diabetic complications.

As used herein the term “diabetes” is a kind of metabolic disease inwhich insulin secretion is insufficient or normal functions are notmade. Diabetes is characterized by increased blood glucose levels thatcause various conditions and syndromes. In the case of diabetes, glucoseis excreted with urine.

As used herein, the term “diabesity” refers to diabetes accompanied byobesity conditions, particularly type 2 diabetes, or obesity conditionsthat generally appear in type 2 diabetic patients. About 80-90% of type2 diabetic patients have obesity conditions and are characterized byinsulin resistances. Proper exercise, diet therapy and drug therapy canprevent diabesity and alleviate the conditions of diabesity. In thepresent invention, diabesity may mean one resulting from obesity.

As used herein, the term

iabetic complications? refers to various pathological conditionsoccurring in the body due to hyperglycemic conditions maintained for along period of time. Examples of diabetic complications include, but arenot limited to, retinopathy, renal dysfunction, neuropathy, strokecaused by vascular disorder, kidney or heart diseases, diabetic footulcer, and cardiovascular disease. If a hyperglycemic condition ismaintained for a long period of time, it leads to various pathologicalconditions in the body. Typically, it increases the risk of retinopathy,renal dysfunction, neuropathy, stroke caused by vascular disorder,kidney or heart diseases, diabetic foot ulcer, and cardiovasculardisease. Thus, to prevent diabetic complications, the effective controlof blood glucose levels is essential.

Accordingly, the pharmaceutical composition of the present invention canbe used for the prevention or treatment of diabetes, diabesity ordiabetic complications.

In an example of the present invention, a long-acting oxyntomodulinanalog conjugate of the present invention was prepared by covalentlylinking the oxyntomodulin analog of the present invention to animmunoglobulin Fc region by polyethylene glycol, and the preparedconjugate was administered to a mouse model with obesity induced byhigh-fat diet and a mouse model with diabetes induced by a mutation inthe leptin receptor. As a result, it was shown that the body weight andfeed intake of the group administered with the long-acting oxyntomodulinanalog conjugate of the present invention significantly decreased thoseof the obesity-induced animal model (FIG. 1) and that the blood glucoselevel significantly decreased (FIG. 2). In addition, the long-actingoxyntomodulin analog conjugate of the present invention showed a bloodglucose lowering effect equal to or higher than VICTOZA® a commerciallyavailable long-acting GLP-1 analog (FIG. 2).

In an example of the present invention, a long-acting oxyntomodulinanalog conjugate was prepared by covalently linking the oxyntomodulinanalog of the present invention to an immunoglobulin Fc region, and theprepared conjugate was administered to a mouse model with diabetesinduced by a mutation in the leptin receptor. As a result, it was shownthat, in the group administered with the long-acting oxyntomodulinanalog conjugate, an increase in the body weight was significantlyinhibited compared to that of the control group (FIG. 3), and the bloodglucose level significantly decreased (FIG. 4), and also the conjugateshowed a superior blood glucose lowering effect compared to VICTOZA® acommercially available long-acting GLP-1 analog (FIG. 4).

In other words, the oxyntomodulin analog according to the presentinvention functions to induce the expansion of beta-cells in vivo toincrease insulin secretion, thereby improving the ability to controlblood glucose levels. In addition, the oxyntomodulin analog according tothe present invention induces a decrease in body weight to improveinsulin sensitivity and prevent the development of cardiovasculardiseases, including arteriosclerosis, hyperlipidemia and hypertension,which can be developed die to insulin resistance. Accordingly, theoxyntomodulin analog of the present invention can be effectively used asan agent for treating diabetes, diabesity and diabetic complications.Additionally, the conjugate of the present invention has a high abilityto activate the GLP-1 receptor and the glucagon receptor, compared tonative oxyntomodulin, and it shows an increased blood half-life in vivodue to the Fc region bound thereto, and thus the activity thereof can bemaintained in vivo for an extended period of time.

The composition of the present invention may be a pharmaceuticalcomposition.

The pharmaceutical composition of the present invention may furthercomprise a pharmaceutical agent showing a preventive or therapeuticeffect against diabetes, diabesity or diabetic complications. In orderto administer the oxyntomodulin analog of the present invention incombination with a pharmaceutical agent known as a therapeutic agentagainst diabetes, diabesity or diabetic complications, the compositionof the present invention may further comprise this known pharmaceuticalagent.

Thus, the composition of the present invention may be used alone oradministered in combination with other drugs in order to prevent ortreat diabetes, diabesity or diabetic complications.

As used herein, the term “administration” means introducing a givenmaterial into a patient by any appropriate method. The analog of thepresent invention may be administered by any general route, as long asit can reach a target tissue. Specifically, the analog of the presentinvention may be administered intraperitoneally, intravenously,intramuscularly, subcutaneously, intradermally, orally, locally,intranasally, intrapulmonarily or intrarectally, but is not limitedthereto. However, because the peptide is digested when beingadministered orally, the oral composition is preferably formulated sothat the active ingredient is coated or protected from degradation inthe stomach. Preferably, the composition of the present invention may beadministered in an injectable form. In addition, the pharmaceuticalcomposition of the present invention may be administered using anysystem capable of delivering the active ingredient to target cells.

The pharmaceutical composition comprising the oxyntomodulin analog ofthe present invention may further comprise a pharmaceutically acceptablecarrier. For oral administration, pharmaceutically acceptable carriersinclude a binder, a lubricant, a disintegrant, an excipient, asolubilizer, a dispersing agent, a stabilizer, a suspending agent, acolorant, and a flavoring agent. For injectable preparations,pharmaceutically acceptable carriers include a buffer, a preservative,an analgesic, a solubilizer, an isotonic agent, and a stabilizer. Fortopical administration, pharmaceutically acceptable carriers include abase, an excipient, a lubricant, and a preservative. The pharmaceuticalcomposition of the present invention may be formulated in various dosageforms using the aforementioned pharmaceutically acceptable carriers. Forexample, for oral administration, the pharmaceutical composition may beformulated into tablets, troches, capsules, elixirs, suspensions,syrups, wafers or the like. For injectable preparations, thepharmaceutical composition may be provided in the form of a unit dosageampoule or a multiple dosage container. In addition, the pharmaceuticalcomposition may also be formulated into solutions, suspensions, tablets,pills, capsules and sustained-release preparations.

Meanwhile, examples of the carrier, excipient and diluent suitable forformulation include lactose, dextrose, sucrose, sorbitol, mannitol,xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin,calcium phosphate, calcium silicate, cellulose, methylcellulose,microcrystalline cellulose, polyvinylpyrrolidone, water,methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearateand mineral oils. In addition, the pharmaceutical composition of thepresent invention may further include fillers, anti-coagulating agents,lubricants, wetting agents, flavors, preservative and the like.

The dose of the pharmaceutical composition of the present invention isdetermined according to the kind of active ingredient together withvarious factors such as the disease to be treated, the route ofadministration, the patient's age, sex and weight, and the severity ofthe disease. The pharmaceutical composition of the present invention hasa long in vivo half-life and excellent bioavailability, and thus thenumber and frequency of administration of the pharmaceutical compositioncan be significantly reduced.

In another aspect, the present invention provides a method forpreventing or treating diabetes, diabesity or diabetic complications,the method comprising administering a pharmaceutically effective amountof the oxyntomodulin analog to a subject.

Herein, the oxyntomodulin analog, diabetes, diabesity and diabeticcomplications are as defined above.

As used herein, the term “subject” refers to a subject suspected ofhaving diabetes, diabesity or diabetic complications. Specifically, theterm means mammals, including humans, rats and domestic animals, whichhave or are at the risk of developing the above disease. In addition,the subject may be any subject that can be treated by the oxyntomodulinanalog of the present invention.

The therapeutic method of the present invention may compriseadministering a pharmaceutically effective amount of the pharmaceuticalcomposition comprising the conjugate. The total daily dose of thecomposition can be determined through appropriate medical judgment by aphysician, and the composition may be administered once or severaltimes. However, in view of the purpose of the present invention, thespecific therapeutically effective dose of the composition for anyparticular patient may vary depending on various factors well known inthe medical field, including the kind and degree of response to beachieved, concrete compositions according to whether other agents areused therewith or not, the patient's age, body weight, health condition,sex and diet, the time and route of administration, the secretion rateof the composition, the duration of treatment, other drugs used incombination or coincident with the composition of the present invention,and other factors known in the medical field.

In still another aspect, the present invention provides the use of theoxyntomodulin analog of the present invention in the preparation of amedicament for preventing or treating diabetes, diabesity or diabeticcomplications.

In yet another aspect, the present invention provides a method forpreparing the oxyntomodulin analog conjugate.

The preparation method of the present invention may comprise the stepsof: (1) covalently linking a non-peptidyl polymer having a reactivealdehyde, maleimide or succinimide group at both ends to the amine orthiol group of an oxyntomodulin analog peptide; (2) separating aconjugate comprising the oxyntomodulin analog peptide, having thenon-peptidyl polymer covalently linked thereto at positions other thanthe amino terminal end, from the reaction mixture of step (1); and (3)covalently linking an immunoglobulin Fc region to the other end of thelinked non-peptidyl polymer of the separated conjugate, therebyproducing a peptide conjugate comprising the immunoglobulin Fc regionand the oxyntomodulin analog peptide, linked to both ends of thenon-peptidyl polymer, respectively.

More specifically, the preparation method may comprise the steps of: (1)covalently linking a non-peptidyl polymer, having a reactive aldehydegroup and a reactive maleimide group at each end thereof, to thecysteine residue of an oxyntomodulin analog; (2) separating a conjugatecomprising the oxyntomodulin analog, having the non-peptidyl polymercovalently linked to the cysteine residue, from the reaction mixture ofstep (1); and (3) covalently linking an immunoglobulin Fc region to theother end of the linked non-peptidyl polymer of the separated conjugate,thereby producing a peptide conjugate comprising the immunoglobulin Fcregion and the oxyntomodulin analog, linked to both ends of thenon-peptidyl polymer, respectively.

MODE FOR INVENTION

Hereinafter, the present invention will be described in further detailwith reference to examples. It is to be understood, however, that theseexamples are for illustrative purposes only and are not intended tolimit the scope of the present invention.

Example 1 Production of Cell Line for In Vitro Activation Example 1-1Production of Cell Line Showing cAMP Response to GLP-1

Using a portion corresponding to the ORF (open reading frame) of cDNA(OriGene Technologies, Inc. USA) of the human GLP-1 receptor gene as atemplate, PCR was performed using reverse and forward primers includinga HindIII cleavage site and an EcoRI cleavage site, respectively,thereby obtaining a PCR product.

Forward primer: (SEQ ID NO: 50) 5′-CCCGGCCCCCGCGGCCGCTATTCGAAATAC-3′Reverse primer: SEQ ID NO: 51) 5′-GAACGGTCCGGAGGACGTCGACTCTTAAGATAG-3′

The PCR product was cloned into the known animal cell expression vectorx0GC/dhfr, thereby constructing the recombinant vector x0GC/GLP-1R.

The recombinant vector x0GC/GLP-1R was introduced into a CHO DG44 cellline, cultured in DMEM/F12 (10% FBS) medium, using lipofectamine(Invitrogene, USA), to obtain a transformant. The transformant wasincubated in a selective medium containing 1 mg/mL G418 and 10 nMmethotraxate, and monoclonal cell lines were selected therefrom. Then, acell line showing a good concentration-dependent cAMP response to GLP-1was finally selected from the monoclonal cell lines.

Example 1-2 Production of Cell Line Showing cAMP Response to Glucagon

Using a portion corresponding to the ORF (open reading frame) of cDNA(OriGene Technologies, Inc. USA) of the human glucagon receptor gene asa template, PCR was performed using reverse and forward primersincluding an EcoRI cleavage site and a XhoI cleavage site, respectively,thereby obtaining a PCR product.

Forward primer: (SEQ ID NO: 52) 5′-CAGCGACACCGACCGTCCCCCCGTACTTAAGGCC-3′Reverse Primer: (SEQ ID NO: 53) 5′-CTAACCGACTCTCGGGGAAGACTGAGCTCGCC-3′

The PCR product was cloned into the known animal cell expression vectorx0GC/dhfr, thereby constructing the recombinant vector x0GC/GCGR.

The recombinant vector x0GC/GCGR was introduced into a CHO DG44 cellline, cultured in DMEM/F12 (10% FBS) medium, using lipofectamine(Invitrogene, USA), to obtain a transformant. The transformant wasincubated in a selective medium containing 1 mg/mL G418 and 10 nMmethotraxate, and monoclonal cell lines were selected therefrom. Then, acell line showing a good concentration-dependent cAMP response toglucagon was finally selected from the monoclonal cell lines.

Example 2 In Vitro Activity of Oxyntomodulin Analogs Example 2-1Synthesis of Oxyntomodulin Analogs

In order to measure the in vitro activities of oxyntomodulin analogs,oxyntomodulin analogs having the amino acid sequences shown in Table 1below were synthesized.

Oxyntomodulin and oxyntomodulin analogs SEQ ID NO Sequence SEQ ID NO: 1HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRN NIA SEQ ID NO: 2CA-SQGTFTSDYSKYLDEEAVRLFIEWLMNTKRN RNNIA SEQ ID NO: 3CA-SQGTFTSDYSKYLDERRAQDFVAWLKNTGPS SGAPPPS SEQ ID NO: 4CA-GQGTFTSDYSRYLEEEAVRLFIEWLKNGGPS SGAPPPS SEQ ID NO: 5CA-GQGTFTSDYSRQMEEEAVRLFIEWLKNGGPS SGAPPPS SEQ ID NO: 6CA-GEGTFTSDLSRQMEEEAVRLFIEWAAHSQGT FTSDYSKYLD SEQ ID NO: 7CA-SQGTFTSDYSRYLDEEAVRLFIEWLMNTK SEQ ID NO: 8CA-SQGTFTSDLSRQLEEEAVRLFIEWLMNK SEQ ID NO: 9CA-GQGTFTSDYSRYLDEEAVXLFIEWLMNTKRN RNNIA SEQ ID NO: 10CA-SQGTFTSDYSRQMEEEAVRLFIEWLMNGGPS SGAPPPSK SEQ ID NO: 11CA-GEGTFTSDLSRQMEEEAVRLFIEWAAHSQGT FTSDYSRYLDK SEQ ID NO: 12CA-SQGTFTSDYSRYLDGGGHGEGTFTSDLSKQM EEEAVK SEQ ID NO: 13CA-SQGTFTSDYSRYLDXEAVXLFIEWLMNTK SEQ ID NO: 14CA-GQGTFTSDYSRYLDEEAVXLFIXWLMNTKRN RNNIA SEQ ID NO: 15CA-GQGTFTSDYSRYLDEEAVRLFIXWLMNTKRN RNNIA SEQ ID NO: 16CA-SQGTFTSDLSRQLEGGGHSQGTFTSDLSRQL EK SEQ ID NO: 17CA-SQGTFTSDYSRYLDEEAVRLFIEWIRNTKRN RNNIA SEQ ID NO: 18CA-SQGTFTSDYSRYLDEEAVRLFIEWIRNGGPS SGAPPPSK SEQ ID NO: 19CA-SQGTFTSDYSRYLD E EAV K LFIEWIRNTKRN RNNIA SEQ ID NO: 20CA-SQGTFTSDYSRYLD E EAV K LFIEWIRNGGPS SGAPPPSK SEQ ID NO: 21CA-SQGTFTSDYSRQLEEEAVRLFIEWVRNTKRN RNNIA SEQ ID NO: 22 DA-SQGTFTSDYSKYLDE KRA K EFVQWLMNTK SEQ ID NO: 23 HAibQGTFTSDYSKYLDEKRAKEFVCWLMNTSEQ ID NO: 24 HAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC SEQ ID NO: 25HAibQGTFTSDYSKYLD E KRA K EFVQWLMNTC SEQ ID NO: 26 HAibQGTFTSDYS K YLD EKRAKEFVQWLMNTC SEQ ID NO: 27 HAibQGTFTSDYSKYLD E QAA K EFICWLMNTSEQ ID NO: 28 HAibQGTFTSDYSKYLDEKRAKEFVQWLMNT SEQ ID NO: 29H(d)SQGTFTSDYSKYLDSRRAQDFVQWLMNTKR NRNNIA SEQ ID NO: 30CA-SQGTFTSDYSKYLDSRRAQDFVQWLMNTKRN RNNIA SEQ ID NO: 31CA-(d)SQGTFTSDYSKYLDSRRAQDFVQWLMNT KRNRNNIA SEQ ID NO: 32CA-AibQGTFTSDYSKYLDEKRAKEFVQWLMNTC SEQ ID NO: 33HAibQGTFTSDYAKYLDEKRAKEFVQWLMNTC SEQ ID NO: 34YAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC

In Table 1 above, the amino acids indicated by the bold letters meanring formation, and the amino acids indicated by X meanalpha-methyl-glutamic acids that are non-native amino acids. Inaddition, CA indicates 4-imidazoacetyl, DA indicates desamino-histidyl,and (d)S indicates d-serine.

Example 2-2 Measurement of In Vitro Activities of Oxyntomodulin Analogs

In order to measure the effects of the peptides prepared in Example 2-1above, the in vitro activities of the peptides in cells were measuredusing the transformants prepared in Examples 1-1 and 1-2.

Each of the transformants was transformed so as to express each of humanGLP-1 receptor and glucagon receptor genes in CHO (Chinese hamsterovary) and was suitable for measuring the activities of GLP-1 andglucagon. Thus, the activity of each of the oxyntomodulin analogs wasmeasured using each of the transformants.

Specifically, each of the transformants was subcultured twice or threetimes a week, and the cells were dispensed into each well of a 96-wellplate at a density of 1×10⁵ cells/well and cultured for 24 hours.

The cultured cells were washed with KRB buffer, suspended in 40 ml of 1mM IBMX-containing KRB buffer, and then allowed to stand at roomtemperature for 5 minutes. Each of oxyntomodulin (SEQ ID NO: 1) and theoxyntomodulin analogs (SEQ ID NOS: 2-6, 8, 10-13, 17, 18, 23-25, 27, 28and 32-34) was serially diluted by five-fold from 1000 nM to 0.02 nM,and 40 ml of each of the dilutions was added to the cells, which werethen incubated in a CO₂ incubator at 37° C. for 1 hour. Then, 20 ml ofcell lysis buffer was added to lyse the cells, and the concentration ofcAMP in each of the cell lysates was measured using a cAMP assay kit(Molecular Device, USA). From the results of the measurement, EC₅₀values were calculated and compared with each other (Table 2).

TABLE 2 Comparison of in vitro activities of GLP-1 receptor and glucagonreceptor between oxyntomodulin analogs EC₅₀ (nM) SEQ ID NO CHO/GLP-1RCHO/GCGR SEQ ID NO: 1 50-210 10-43 SEQ ID NO: 2 51.8 12.8 SEQ ID NO:3 >1,000 637.7 SEQ ID NO: 4 5.5 >1,000 SEQ ID NO: 5 5.9 >1,000 SEQ IDNO: 6 500.1 >1,000 SEQ ID NO: 8 419.6 >1,000 SEQ ID NO: 10 >1,000 >1,000SEQ ID NO: 11 >1,000 >1,000 SEQ ID NO: 12 >1,000 >1,000 SEQ ID NO:13 >1,000 >1,000 SEQ ID NO: 17 97.9 >1,000 SEQ ID NO: 18 96.3 >1,000 SEQID NO: 23 2.46 5.8 SEQ ID NO: 24 1.43 6.95 SEQ ID NO: 25 1.9 1.3 SEQ IDNO: 27 2.8-5.5 3.1-5.6 SEQ ID NO: 28 3.1 0.3 SEQ ID NO: 32 41.3 17.7 SEQID NO: 33 2.2 80.2 SEQ ID NO: 34 12.5 1.04

As can be seen in Table 2 above, the oxyntomodulin analogs showedexcellent in vitro GLP-1 and glucagon receptor activities compared tothe oxyntomodulin of SEQ ID NO: 1. Oxyntomodulin is known to have theeffect of treating obesity, hyperlipidemia, fatty liver disease orarteriosclerosis by activating the GLP-1 receptor and the glucagonreceptor. The oxyntomodulin analogs according to the present inventionhave a high ability to activate the GLP-1 receptor and the glucagonreceptor in vitro, compared to native oxyntomodulin, suggesting thatthese oxyntomodulin analogs are highly effective in treating diabetes,diabesity or diabetic complications, compared to native oxyntomodulin.

Example 3 Preparation of a Conjugate Comprising Oxyntomodulin Analog(SEQ ID NO: 23) and Immunoglobulin Fc (Immunoglobulin Fc-ConjugatedOxyntomodulin Analog 23)

In order to PEGylate MAL-10K-ALD PEG (NOF., Japan) at a cysteine residueat position 24 of the amino acid sequence of the oxyntomodulin analog(SEQ ID NO: 23), the oxyntomodulin analog (SEQ ID NO: 23) andMAL-10K-ALD PEG were allowed to react with each other at a molar ratioof 1:3 at a protein concentration of 3 mg/ml at room temperature for 3hours. The reaction was performed in 50 mM Tris buffer (pH 8.0)containing 1M guanidine. After completion of the reaction, the reactionsolution was applied to SOURCE S under the following conditions, therebypurifying an oxyntomodulin analog mono-PEGylated at the cysteine:column: SOURCE S, flow rate: 2.0 ml/min, gradient: A 0->100% 50 min B(A: 20 mM Na-citrate (pH 3.0)+45% ethanol, B: A+1M KCl).

Then, the purified mono-pegylated oxyntomodulin analog (SEQ ID NO: 23)and an immunoglobulin Fc were allowed to react with each other at amolar ratio of 1:5 at a protein concentration of 20 mg/ml at 4° C. for16 hours. The reaction was performed in 100 mM potassium phosphatebuffer (pH 6.0) containing 20 mM SCB as a reducing agent. Aftercompletion of the reaction, the reaction solution was applied to aSOURCE purification column (column: SOURCE 15Q, flow rate: 2.0 ml/min,gradient: A 0->4% 1 min, B->20% 80 min B (A: 20 mM Tris-HCl, pH 7.5, B:A+1M NaCl)) and a Source ISO column (column: SOURCE ISO, flow rate: 2.0ml/min, gradient: B 0->100% 100 min A, (A: 20 mM Tris-HCl, pH 7.5, B:A+1.1M AS)), thereby purifying a conjugate comprising the oxyntomodulinanalog (SEQ ID NO: 23) and the immunoglobulin Fc.

Example 4 Preparation of a Conjugate Comprising Oxyntomodulin Analog(SEQ ID NO: 25) and Immunoglobulin Fc (Immunoglobulin Fc-ConjugatedOxyntomodulin Analog 25)

In order to PEGylate MAL-10K-ALD PEG at a cysteine residue at position30 of the amino acid sequence of an oxyntomodulin analog (SEQ ID NO:25), the oxyntomodulin analog (SEQ ID NO: 25) and MAL-10K-ALD PEG wereallowed to react with each other at a molar ratio of 1:3 at a proteinconcentration of 3 mg/ml at room temperature for 3 hours. The reactionwas performed in 50 mM Tris buffer (pH 8.0) containing 1M guanidine.After completion of the reaction, the reaction solution was applied toSOURCE S under the following conditions, thereby purifying anoxyntomodulin analog mono-PEGylated at the cysteine: column: SOURCE S,flow rate: 2.0 ml/min, gradient: A 0->100% 50 min B (A: 20 mM Na-citrate(pH 3.0)+45% ethanol, B: A+1M KCl).

Then, the purified mono-PEGylated oxyntomodulin analog (SEQ ID NO: 25)and an immunoglobulin Fc were allowed to react with each other at amolar ratio of 1:5 at a protein concentration of 20 mg/ml at 4° C. for16 hours. The reaction was performed in 100 mM potassium phosphatebuffer (pH 6.0) containing 20 mM SCB as a reducing agent. Aftercompletion of the reaction, the reaction solution was applied to aSOURCE 15Q column (column: SOURCE 15Q, flow rate: 2.0 ml/min, gradient:A 0->4% 1 min B->20% 80 min B (A: 20 mM Tris-HCl (pH 7.5), B: A+1MNaCl)) and a Source ISO column (column: SOURCE ISO, flow rate: 2.0ml/min, flow rate: B 0->100% 100 min A (A: 20 mM Tris-HCl (pH 7.5), B:A+1.1M AS)), thereby purifying a conjugate comprising the oxyntomodulinanalog (SEQ ID NO: 25) and the immunoglobulin Fc.

Example 5 Preparation of a Conjugate Comprising Oxyntomodulin Analog(SEQ ID NO: 27) and Immunoglobulin Fc (Immunoglobulin Fc-ConjugatedOxyntomodulin Analog 27)

In order to PEGylate MAL-10K-ALD PEG at a cysteine residue at position30 of the amino acid sequence of an oxyntomodulin analog (SEQ ID NO:27), the oxyntomodulin analog (SEQ ID NO: 27) and MAL-10K-ALD PEG wereallowed to react with each other at a molar ratio of 1:3 at a proteinconcentration of 3 mg/ml at room temperature for 3 hours. The reactionwas performed in 50 mM Tris buffer (pH 8.0) containing 1M guanidine.After completion of the reaction, the reaction solution was applied toSOURCE S under the following conditions, thereby obtaining anoxyntomodulin analog mono-PEGylated at the cysteine: column: SOURCE S,flow rate: 2.0 ml/min, gradient: A 0->100% 50 min B (A: 20 mM Na-citrate(pH 3.0)+45% ethanol, B: A+1M KCl).

Then, the purified mono-PEGylated oxyntomodulin analog (SEQ ID NO: 27)and an immunoglobulin Fc were allowed to react with each other at amolar ratio of 1:5 at a protein concentration of 20 mg/ml at 4° C. for16 hours. The reaction was performed in 100 mM potassium phosphatebuffer (pH 6.0) containing 20 mM SCB as a reducing agent. Aftercompletion of the reaction, the reaction solution was applied to aSOURCE 15Q column (column: SOURCE 15Q, flow rate: 2.0 ml/min, gradient:A 0->4% 1 min B->20% 80 min B (A: 20 mM Tris-HCl (pH 7.5), B: A+1MNaCl)) and a Source ISO column (column: SOURCE ISO, flow rate: 2.0ml/min, gradient: B 0->100% 100 min A (A: 20 mM Tris-HCl (pH 7.5), B:A+1.1M AS)), thereby purifying a conjugate comprising the oxyntomodulinanalog (SEQ ID NO: 27) and the immunoglobulin Fc.

Example 6 Effects of Long-Acting Oxyntomodulin Analog on Reduction inthe Body Weight and Blood Glucose Level of High-Fat Diet-Induced Obesity(HF DIO) Mice Example 6-1 Experimental Method

6-Week-old mice (C57BL/6, 120-130 g) were purchased from OrientBIO(Korea). The purchased C57BL/6 mice are animals that are widely used instudies on obesity and diabetes, because obesity therein can berelatively easily induced by high-fat diet. HF DIO mice are rodents thatare frequently used in diabetes studies, and naturally show obesity anddiabetic conditions similar to those of humans as a result oftransplanting a high-fat diet into the organ without geneticmanipulation, unlike db/db mice with diabetes induced by a mutation inthe leptin receptor. For this reason, in the present invention, theseanimals were used to examine the effects of the composition of thepresent invention on reductions in body weight and blood glucose levelsin diabesity.

The animals were allowed access to a high-fat diet (60% Kcal from fatdiet, D12492; Research Diets Inc.) sterilized by UV irradiation. Also,the animals were allowed access to filtered and UV-sterilized tap waterusing water bottles. The animals were kept in a breeding chambersatisfying GLP standards under a 12-hr light/12-hr dark cycle (lighting:am 6 to pm 6), and all the experimental procedures were performedaccording to the standard guideline for animal experiments. Drugadministration was started after 26 weeks of obesity induction, and theanimals were divided into five groups (n=6) as shown in Table 3 below.

TABLE 3 Methods of Groups Drugs administered administration HF Vehicle(PBS) S.C. Once a D10-induced week group HF VICTOZA ® 100 nmol/kg S.C.Once a D10-induced SEQ ID NO: 25-Fc conjugate 1 nmol/kg day plus SEQ IDNO: 25-Fc conjugate 3 nmol/kg S.C. Once a drug- SEQ ID NO: 25-Fcconjugate 5 nmol/kg week administered groups

Specifically, group 1 (HF DIO-induced group, control group) was fed withhigh-fat feed and administered subcutaneously with 5 ml/kg (injectionvolume) of Dulbecco's phosphate buffered saline (DPBS, Sigma) once ormore a week. For a blood glucose tolerance test, group 1 wasadministered subcutaneously with Dulbecco's phosphate buffered saline(DPBS, Sigma) at 24 hours before the test and fasted for 16 hours. Theblood was collected from the tail portion to measure the fasting glucoselevel, and the blood glucose levels at 15 min, 30 min, 60 min, 90 minand 120 min after intra-abdominal administration of 1 g/kg of glucosewere measured. Group 2 (HF DIO-induced and 100 nmol/kg VICTOZA®administered group) was fed with high-fat diet to induce obesity andhyperglycemia, and then administered once a day subcutaneously with 5ml/kg (injection volume) of commercially available VICTOZA®(GSK). For ablood glucose tolerance test, group 2 was fasted for 16 hours before thetest and administered subcutaneously with 100 nmol/kg of VICTOZA® at 4hours before the test. The blood was collected from the tail portion tomeasure the fasting glucose level, and the blood glucose levels at 15min, 30 min, 60 min, 90 min and 120 min after intra-abdominaladministration of 1 g/kg of glucose were measured.

Group 3 (HF DIO-induced and 1 nmol/kg SEQ ID NO: 25-Fcconjugate-administered group) was fed with high-fat feed to induceobesity and hyperglycemia, and then administered subcutaneously once aweek with 1 nmol/kg (injection volume of 5 ml/kg) of the SEQ ID NO:25-Fc conjugate prepared in Example 4. For a blood glucose tolerancetest, group 3 was fasted for 24 hours before the test and administeredsubcutaneously with 1 nmol/kg of the SEQ ID NO: 25-Fc conjugate at 24hours before the test and fasted for 16 hours. The blood was collectedfrom the tail portion to measure the fasting glucose level, and theblood glucose levels at 15 min, 30 min, 60 min, 90 min and 120 min afterintra-abdominal administration of 1 g/kg of glucose were measured.

Group 4 (HF DIO-induced and 3 nmol/kg SEQ ID NO: 25-Fcconjugate-administered group) was fed with high-fat feed to induceobesity and hyperglycemia, and then administered subcutaneously once aweek with 3 nmol/kg (injection volume of 5 ml/kg) of the SEQ ID NO:25-Fc conjugate prepared in Example 4. For a blood glucose tolerancetest, group 3 was fasted for 24 hours before the test and administeredsubcutaneously with 3 nmol/kg of the SEQ ID NO: 25-Fc conjugate at 24hours before the test and fasted for 16 hours. The blood was collectedfrom the tail portion to measure the fasting glucose level, and theblood glucose levels at 15 min, 30 min, 60 min, 90 min and 120 min afterintra-abdominal administration of 1 g/kg of glucose were measured.

Group 5 (HF DIO-induced and 5 nmol/kg SEQ ID NO: 25-Fcconjugate-administered group) was fed with high-fat feed to induceobesity and hyperglycemia, and then administered subcutaneously once aweek with 5 nmol/kg (injection volume of 5 ml/kg) of the SEQ ID NO:25-Fc conjugate prepared in Example 4. For a blood glucose tolerancetest, group 3 was fasted for 24 hours before the test and administeredsubcutaneously with 5 nmol/kg of the SEQ ID NO: 25-Fc conjugate at 24hours before the test and fasted for 16 hours. The blood was collectedfrom the tail portion to measure the fasting glucose level, and theblood glucose levels at 15 min, 30 min, 60 min, 90 min and 120 min afterintra-abdominal administration of 1 g/kg of glucose were measured.

For all the groups (n=6), saline or each drug was administered for 2weeks, and then the effects thereof on reductions in the body weight andthe blood glucose level were analyzed.

Example 6-2 Effects of Long-Acting Oxyntomodulin Analog on Reductions inBody Weight and Blood Glucose Level of High-Fat Diet-Induced Obesity (HFDIO) Mice which are Stable Obesity Models

In order to examine the effect of the long-lasting oxyntomodulin analogof the present invention on a reduction in the blood glucose level ofhigh-fat diet-induced (for 26 weeks) obesity (HF DIO) mice which arestable obesity models, the DIO mice classified in Example 6-1 wereadministered subcutaneously with the long-acting oxyntomodulin analogonce a week for 2 weeks. The body weight and the feed intake weremeasured every day, and the blood was collected from the tail portion ofthe DIO mice at days 0, 3, 7, 10 and 14, and the change in the bloodglucose levels was analyzed using HITACHI 7020. The changes in the bodyweight and the blood glucose level are shown in FIGS. 1 and 2.

FIG. 1 shows the change in the body weight, and FIG. 2 shows the bloodglucose AUC (area under curve). The obtained results were statisticallyprocessed, and the mean values and the standard deviations of the meanvalues were calculated.

In the verification of significance between the groups (n=6), data werestatistically processed using Dunnett's test of one-way ANOVA, and avalue of p<0.05 was considered statistically significant.

Specifically, the results of measurement of the change in the bodyweight indicated that the body weight of the mice with obesity inducedby high-fat diet for 26 weeks did not decrease, whereas, when the micewith obesity were administered with the long-acting oxyntomodulin analog(SEQ ID NO: 25-Fc conjugate), the body weight thereof decreased in adose-dependent manner (FIG. 1).

The results of measurement of the blood glucose level indicated that theblood glucose level of the mice with obesity decreased in adose-dependent manner, when the mice were administered with thelong-acting oxyntomodulin analog (SEQ ID NO: 25-Fc conjugate).Particularly, when the mice with obesity were administered with 5nmol/kg of the long-acting oxyntomodulin analog (SEQ ID NO: 25-Fcconjugate), the blood glucose level thereof significantly decreasedcompared to that of the high-fat diet-induced DIO mice, and the bloodglucose lowering effect of 5 nmol/kg of the long-acting oxyntomodulinanalog (SEQ ID NO: 25-Fc conjugate) was equal to or better than that ofVICTOZA® that is a commercially available drug for treating diabetes(FIG. 2).

From the results of Example 6-2, it was found that the long-actingoxyntomodulin analog conjugate of the present invention, which comprisesthe oxyntomodulin analog covalently linked to the immunoglobulin Fcregion by PEG, reduced the body weight and blood glucose level of thehigh-fat diet-induced obesity (HF DIO) mice, suggesting that it can beeffectively used for the treatment of diabetes, diabesity or relateddiseases.

Example 7 Effects of Long-Acting Oxyntomodulin Analog on Reductions inthe Body Weight and Blood Glucose Level of Db/Db Mice with DiabetesInduced by Mutation in Leptin Receptor Example 7-1 Experimental Method

7-week old male BKS.Cg−+Lepr^(db)/+Lepr^(db)/OlaHsd mice (25±3 g, HarlanU.S.A) were purchased from Doo Yeol Biotech (Korea).BKS.Cg−+Lepr^(db)/+Lepr^(db)/OlaHsd mice (hereinafter referred to asdb/db mice) are rodents that are most frequently used in diabetesstudies together with ob/ob mice, and these mice naturally show diabeticconditions similar to those of humans through a mutation in the leptinreceptor. For this reason, in the present invention, these animals wereused to examine the blood glucose lowering effect of the agent of thepresent invention in the development of an agent for treating diabetes.

The purchased animals were acclimated and adapted to the experimentalenvironment for 1 week, and then randomly grouped according to theirglucose levels.

The animals were allowed access to solid feed (Picolab Rodent diet 5053)sterilized by UV irradiation. Also, the animals were allowed access tofiltered and UV-sterilized tap water using water bottles. The animalswere kept in a breeding chamber satisfying GLP standards under a 12-hrlight/12-hr dark cycle (lighting: 6 a.m. to 6 p.m.), and all theexperimental procedures were performed according to the standardguideline for animal experiments. The animals were divided into fourgroups (n=7) and administered with drugs as shown in Table 4 below.

TABLE 4 Methods of Groups Drugs administered administration Controlgroup Vehicle (PBS) S.C. Once a week × 4 Groups VICTOZA ® 60 nmol/kgS.C. Once a day × 28 administered with drugs VICTOZA ® 100 nmol/kg SEQID NO: 23-Fc conjugate S.C. Once a week × 4 15 nmol/kg SEQ ID NO: 25-Fcconjugate 6 nmol/kg

Specifically, group 1 (vehicle), a control group, was administeredsubcutaneously with 5 ml/kg of Dulbecco's phosphate buffered saline(DPBS, Sigma) once a week.

Group 2 (administered with 60 nmol/kg of VICTOZA®, a drug-administeredgroup, was administered subcutaneously once a day with 60 nmol/kg (dosefor diabetes; injection volume of 5 ml/kg) of commercially availableVICTOZA®(GSK).

Group (administered with 100 nmol/kg of VICTOZA®, a drug-administeredgroup, was administered subcutaneously once a day with 100 nmol/kg (dosefor obesity; injection volume of 5 ml/kg) of commercially availableVictoza® (GSK).

Group 4 (15 nmol/kg of SEQ ID NO: 23-Fc conjugate), a drug-administeredgroup, was administered subcutaneously once a week with 15 nmol/kg(injection volume of 5 ml/kg) of the SEQ ID NO: 23-Fc conjugate preparedin Example 4.

Group 5 (6 nmol/kg of SEQ ID NO: 25-Fc conjugate), a drug-administeredgroup, was administered subcutaneously once a week with 6 nmol/kg(injection volume of 5 ml/kg) of the SEQ ID NO: 25-Fc conjugate preparedin Example 4.

For all the groups (n=7), saline or each drug was administered for 4weeks, and then the effects thereof on reductions in the body weight andthe blood glucose level were analyzed.

Example 7-2 Analysis of the Effects of Long-Acting Oxyntomodulin Analogon Reductions in the Body Weight and Blood Glucose Level of Db/Db Micewith Diabetes by Mutation in Leptin Receptor

In order to examine the effect of the long-acting oxyntomodulin analogof the present invention on a reduction in the blood glucose level ofdb/db mice with diabetes induced by a mutation in the leptin receptor,the db/db mice classified in Example 7-1 were administeredsubcutaneously with the long-acting oxyntomodulin analog once a week for4 weeks. The change in the body weight of the mice was measured twice aweek, and the blood was collected from the tail portion of the db/dbmice (every day at weeks 1 and 4, and twice a week at weeks 2 and 3),and the change in the blood glucose level was analyzed using HITACHI7020.

FIG. 3 shows the change in the body weight, and FIG. 4 shows the bloodglucose AUC (area under curve). The obtained results were statisticallyprocessed, and the mean values and the standard deviations of the meanvalues were calculated. In the verification of significance between thegroups (n=6), data were statistically processed using Dunnett's test ofone-way ANOVA, and a value of p<0.05 was considered statisticallysignificant.

Specifically, the results of measurement of the change in the bodyweight indicated that the body weight of the db/db mouse control groupcontinuously increased from the day of start of administration, whereas,when the mice were administered with the long-acting oxyntomodulinanalog (the SEQ ID NO: 23-Fc conjugate or the SEQ ID NO: 25-Fcconjugate), the body weight did not substantially change from the bodyweight measured at the day of state of administration, suggesting thatthe conjugate showed a significant effect of inhibiting the increase inthe body weight (FIG. 3).

The results of measurement of the blood glucose level indicated that,when the mice were administered with the long-acting oxyntomodulinanalog (the SEQ ID NO: 23-Fc conjugate or the SEQ ID NO: 25-Fcconjugate), the blood glucose level significantly decreased compared tothat of the control group. Particularly, administration of 6 nmol/kg ofthe long-acting oxyntomodulin analog (SEQ ID NO: 25-Fc conjugate) showeda blood glucose lowering effect compared to that of VICTOZA® that is acommercially available drug for treating diabetes (FIG. 4).

From the results of Example 7, it was found that the long-actingoxyntomodulin analog of the present invention, which comprises theoxyntomodulin analog covalently linked to the immunoglobulin Fc regionby PEG, significantly reduced the blood glucose level (index ofdiabetes) in the db/db mice with diabetes induced by the mutation in theleptin receptor, compared to the vehicle and VICTOZA® that is being usedas a drug for treating diabetes, suggesting that the long-actingoxyntomodulin analog of the present invention can be very effectivelyused for the treatment of diabetes. In addition, the long-actingoxyntomodulin analog of the present invention showed a significanteffect of inhibiting the increase in the body weight, suggesting that itcan reduce diabetic cardiovascular complications.

From the results of Examples 6 and 7, it was seen that the long-lastingoxyntomodulin analog conjugate of the present invention showed anexcellent blood glucose-lowering effect equal to or better than VICTOZA®known to have a blood glucose lowering effect, as well as an excellentbody weight-reducing effect, suggesting that the long-lastingoxyntomodulin analog conjugate of the present invention can beeffectively used as an agent for treating diabetes, diabesity anddiabetic complications, by virtue of its blood level-lowering effect.

Those of ordinary skill in the art will recognize that the presentinvention may be embodied in other specific formed without departingfrom its spirit or essential characteristics. The described embodimentsare to be considered in all respects only as illustrative and notrestrictive. The scope of the present invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within the scope of the present invention.

1. A composition for preventing or treating diabetes, diabesity ordiabetic complications, the composition comprising an oxyntomodulinanalog as an active ingredient.
 2. The composition of claim 1, whereinthe oxyntomodulin analog has an amino acid sequence selected from thegroup consisting of amino acid sequences of SEQ ID NOS: 2 to
 34. 3. Thecomposition of claim 1, wherein the oxyntomodulin analog is in the formof a conjugate with one selected from the group consisting of animmunoglobulin fragment, an antibody, elastin, albumin and fibronectin.4. The composition of claim 3, wherein the conjugate is a conjugate inwhich the oxyntomodulin analog having the amino acid sequence selectedfrom the group consisting of the amino acid sequences of SEQ ID NOS: 2to 34 is linked to an immunoglobulin Fc region through a non-peptidylpolymer.
 5. The composition of claim 4, wherein the non-peptidyl polymeris selected from the group consisting of polyethylene glycol,polypropylene glycol, an ethylene glycol/propylene glycol copolymer,polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran,polyvinyl ethyl ether, PLA (polylactic acid), PLGA (polylactic-glycolicacid), lipid polymers, chitins, hyaluronic acid, and combinationsthereof.
 6. The composition of claim 4, wherein each end of thenon-peptidyl polymer is linked to the immunoglobulin Fc region and theamine group or thiol group of the oxyntomodulin, respectively.
 7. Thecomposition of claim 1, further comprising a pharmaceutical agentshowing preventive or therapeutic effects against diabetes, diabesity ordiabetic complications.
 8. The composition of claim 1, wherein thediabetes is insulin-dependent type 1 diabetes or insulin-independenttype 2 diabetes.
 9. The composition of claim 1, wherein the diabesityresults from obesity.
 10. A method for treating diabetes, diabesity ordiabetic complications, the method comprising administering apharmaceutically effective amount of an oxyntomodulin analog to asubject.